Chagas disease, a neglected tropical disease (NTD) caused by the flagellated protozoan Trypanosoma cruzi (T. cruzi), is a major public health problem. It was initially restricted to Latin America, ...but it is now expanding globally. Host and pathogen interactions are crucial in the establishment of disease, and since 1970, it has been known that eukaryotic cells release extracellular vesicles (EVs), which in turn have an important role in intercellular communication in physiological and pathological conditions. Our study proposed to characterize and compare circulating EVs isolated from the plasma of chronic Chagas disease (CCD) patients and controls. For this, peripheral blood was collected from patients and controls, and mononuclear cells (PBMCs) were isolated and stimulated with parasite EVs, showing that patient cells released fewer EVs than control cells. Then, after plasma separation followed by EV total shedding enrichment, the samples were subjected to ultracentrifugation to isolate the circulating EVs, which then had their size and concentration characterized by nanoparticle tracking analysis (NTA). This showed that patients had a lower concentration of circulating EVs while there were no differences in size, corroborating the in vitro data. Additionally, circulating EVs were incubated with THP-1 cells (macrophages) that, after the interaction, had their supernatant analyzed by ELISA for cytokine detection. In relation to their ability to induce cytokine production, the CCD patient EVs were able to induce a differential production of IFN-γ and IL-17 in relation to controls, with differences being more evident in earlier/less severe stages of the disease. In summary, a decreased concentration of circulating EVs associated with differential activation of the immunological system in patients with CCD is related to parasite persistence and the establishment of chronic disease. It is also a potential biomarker for monitoring disease progression.
Extracellular vesicles (EVs) are lipid bilayer envelopes that encapsulate cell-specific cargo, rendering them promising biomarkers for diverse diseases. Chagas disease, caused by the parasite
, poses ...a significant global health burden, transcending its initial epicenter in Latin America to affect individuals in Europe, Asia, and North America. In this study, we aimed to characterize circulating EVs derived from patients with chronic Chagas disease (CCD) experiencing a reactivation of acute symptoms. Blood samples collected in EDTA were processed to isolate plasma and subsequently subjected to ultracentrifugation for particle isolation and purification. The EVs were characterized using a nanoparticle tracking analysis and enzyme-linked immunosorbent assay (ELISA). Our findings revealed distinctive differences in the size, concentration, and composition of EVs between immunosuppressed patients and those with CCD. Importantly, these EVs play a critical role in the pathophysiology of Chagas disease and demonstrate significant potential as biomarkers in the chronic phase of the disease. Overall, our findings support the potential utility of the CL-ELISA assay as a specific sensitive tool for detecting circulating EVs in chronic Chagasic patients, particularly those with recurrent infection following an immunosuppressive treatment or with concurrent HIV and Chagas disease. Further investigations are warranted to identify and validate the specific antigens or biomarkers responsible for the observed reactivity in these patient groups, which may have implications for diagnosis, the monitoring of treatment, and prognosis.
Ergosterol biosynthesis inhibitors, such as posaconazole and ravuconazole, have been proposed as drug candidates for Chagas disease, a neglected infectious tropical disease caused by the protozoan ...parasite Trypanosoma cruzi. To understand better the mechanism of action and resistance to these inhibitors, a clone of the T. cruzi Y strain was cultured under intermittent and increasing concentrations of ravuconazole until phenotypic stability was achieved. The ravuconazole-selected clone exhibited loss in fitness in vitro when compared to the wild-type parental clone, as observed in reduced invasion capacity and slowed population growth in both mammalian and insect stages of the parasite. In drug activity assays, the resistant clone was above 300-fold more tolerant to ravuconazole than the sensitive parental clone, when the half-maximum effective concentration (EC50) was considered. The resistant clones also showed reduced virulence in vivo, when compared to parental sensitive clones. Cross-resistance to posaconazole and other CYP51 inhibitors, but not to other antichagasic drugs that act independently of CYP51, such as benznidazole and nifurtimox, was also observed. A novel amino acid residue change, T297M, was found in the TcCYP51 gene in the resistant but not in the sensitive clones. The structural effects of the T297M, and of the previously described P355S residue changes, were modelled to understand their impact on interaction with CYP51 inhibitors.
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•A ravuconazole-resistant T. cruzi clone presented reduced in vitro and in vivo fitness.•The ravuconazole-resistant clone presented cross-resistance to other CYP51 inhibitors.•There was no cross-resistance to benznidazole and nifurtimox.•Resistance is associated with a novel structural mutation in the TcCYP51 protein.
We analyzed the frequency, viability, and genetic characteristics of T. gondii in pork heart samples
Thirty-five fresh pork samples were purchased in a slaughterhouse in Erechim city. The DNA was ...extracted and qPCR was performed. T. gondii genotyping was performed using PCR-RFLP analysis. Positive samples were digested and inoculated in mice for viability analysis.
Our results showed that T. gondii DNA was detected in 25.7% of the pork heart samples and genotyping revealed one new atypical strain. The viability analyses demonstrated that 40% of mice presented clinical signs of T. gondii infection. qPCR was positive in the lung, liver, and brain of mice that presented clinical signs of T. gondii infection. Also, the histopathology analysis showed retinal disorganization, retinal detachment, inflammatory cell infiltration, and fibrosis in the eyes analyzed.
Our findings have shown that pork eat from southern Brazil may contain live T. gondii that could be associated with toxoplasmosis.
Abstract
Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for ...several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite‐parasite and parasite‐host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite‐infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells
O sistema financeiro e o crescimento económico estão muito relacionados, sendo este tema alvo de muitas pesquisas nas últimas décadas. Como temos conhecimento, o sistema financeiro pode ser dividido ...em duas vertentes: mercado de capitais e sistema de crédito bancário. Este estudo tem como finalidade estudar a relação entre mercado de capitais, sistema bancário e crescimento económico para Portugal, utilizando dados trimestrais que estão compreendidos entre 1993 e 2016, que como país europeu é expectável que uma economia mais dependente do sistema bancário. De forma a capturar a questão central do estudo as variáveis testadas foram produto interno bruto, rácio de capitalização do mercado de capitais, rácio do crédito doméstico, investimento e, para variável de controlo é utilizado o índice de preços do consumidor. Após a realização dos testes de raízes unitárias para confirmarmos a ordem de integração das variáveis e a sua análise gráfica concluímos que estas são I(1), sendo que não são co-integradas (Johansen test). Modelo Vector Autoregressive (VAR) é então realizado, bem como, as causalidades de Granger, decomposição da variância e funções impulso-resposta são discutidas no presente estudo. As especificações do VAR revelam normalidade, ausência de auto correlação e de homocedasticidade. Como consequência da entrada para a União Monetária Europeia, ocorrendo a substituição física da moeda, revela-se uma mudança de regime económico, mas também a grande crise for provada. Finalmente foi encontrada uma evidência bidirecional nas causalidades de Granger entre mercado de capitais e crescimento económico. Na verdade, o crescimento económico aparenta ser favorável para o sistema de crédito bancário, uma relação unidirecional foi encontrada desde o crescimento económico para o sistema de crédito bancário.
Chronic obstructive pulmonary disease (COPD) is an independent risk factor for osteoporosis. Oral glucocorticoids are deleterious to bone; however, the impact of inhaled glucocorticoids (ICS) remains ...unclear. Our objective was to determine whether ICS contribute to osteoporosis and fragility fractures. Sixty-one COPD patients, 35 current users of ICS and 26 who had never received glucocorticoids, were evaluated for bone mineral density (BMD) and body composition and underwent vertebral fracture assessment (VFA). The risk factors for bone disease considered for analysis were age, gender, ICS use, body mass index (BMI), muscle mass index (MMI), and the Global Initiative for Chronic Obstructive Lung Disease (GOLD) category. The Fracture Risk Assessment Tool (FRAX) calculation tool for the Brazilian population was also employed. The groups did not differ regarding gender, BMI, MMI, GOLD class, lowest values of the BMD
T
-score and
Z
-score, prevalence of osteoporosis, or low BMD for age. Vertebral fractures were identified via VFA in seven patients using ICS and in none of those not receiving glucocorticoids (
p
= 0.02). There was a trend for an association between MMI and osteoporosis (
p
= 0.05) and for a progressive decrease in the BMD
Z
-score according to the COPD severity assessed via the GOLD score (
p
= 0.08). Vertebral fractures were not associated with osteoporosis (
p
= 0.69) or low MMI (
p
= 0.12). The fracture risk was not estimated by FRAX. ICS may lead to bone fragility before a significant decrease in BMD. Low muscle mass and COPD severity may contribute to bone disease.
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